Visible Light-Triggered ROS-Mediated ... - ACS Publications

for infection control. NanoZymes are emerging as a new class of catalytic nanomaterials that mimic the biological action of natural enzymes. Developme...
0 downloads 4 Views 6MB Size
Subscriber access provided by Kaohsiung Medical University

Visible Light-Triggered ROS-Mediated Antibacterial Activity of Peroxidase-Mimic CuO Nanorods Md Nurul Karim, Mandeep Singh, Pabudi Weerathunge, Pengju Bian, Rongkun Zheng, Chaitali Dekiwadia, Taimur Ahmed, Sumeet Walia, Enrico Della Gaspera, Sanjay Singh, Rajesh Ramanathan, and Vipul Bansal ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00153 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

Visible Light-Triggered ROS-Mediated Antibacterial Activity of Peroxidase-Mimic CuO Nanorods Md. Nurul Karim,a‡ Mandeep Singh,a‡ Pabudi Weerathunge,a Pengju Bian,b Rongkun Zheng,b Chaitali Dekiwadia,c Taimur Ahmed,d Sumeet Walia,d Enrico Della Gaspera,e Sanjay Singh,a,f* Rajesh Ramanathan,a* and Vipul Bansala* a

Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory (NBRL) School of Science, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia b

c

School of Physics, The University of Sydney, Sydney, NSW 2006, Australia

RMIT Microscopy and Microanalysis Facility (RMMF), RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia

d

Functional Materials and Microsystems Research Group and Micro Nano Research Facility, School of Engineering, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia e

School of Science, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia

f

Division of Biological & Life Sciences, School of Arts and Sciences, Central Campus, Ahmedabad University, Navrangpura, Ahmedabad 380009, Gujarat, India.

KEYWORDS. peroxidase-mimic; nanozyme; CuO; antibacterial; light; ROS

ACS Paragon Plus Environment

1

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

ABSTRACT. Rapid emergence of antibiotic-resistant bacterial strains warrants new strategies for infection control. NanoZymes are emerging as a new class of catalytic nanomaterials that mimic the biological action of natural enzymes. Development of photoactive NanoZymes offers a promising avenue to use light as a ‘trigger’ to modulate the bacterial activity. Visible light activity is particularly desirable, as it contributes to 44% of the total solar energy. Here we show that the favorable band structure of a CuO nanorod based NanoZyme catalyst (band gap of 1.44 eV) allows visible light to control the antibacterial activity. Photomodulation of the peroxidasemimic activity of CuO nanorods enhances its affinity to H2O2, thereby remarkably accelerating the production of reactive oxygen species (ROS) by 20 times. This photo-induced NanoZymemediated ROS production catalyzes physical damages to the bacterial cells, thereby enhancing the antibacterial performance against Gram negative indicator bacteria Escherichia coli.

ACS Paragon Plus Environment

2

Page 3 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

The rapid emergence of infectious disease outbreaks due to mutating pathogenic strains of bacteria and other microorganisms pose a serious public health risk worldwide.1 Currently, antibiotics act as the primary line of defense through either preventing pathogens from multiplying by a bacteriostatic action, or causing bactericidal effect to directly kill infectious organisms.1-2 A number of other materials including polymers,3 metal ions,4 chemically modified natural compounds,5 and quaternary ammonium compounds,6 have also been explored as antibacterial agents, but not without associated challenges and limitations.5-6 In particular, the extensive use of antibiotics over the past several decades has brought mankind to a stage where efficient management of antibiotic-resistant pathogenic strains has become one of the biggest challenges for our current and future generations.1-2 This has prompted the exploration of new alternative strategies to combat ever-evolving pathogenic bacteria.4, 6-7 Nanoscale materials including metal,8-10 metal oxides,11 and metal-organic semiconductors12 have been explored as a viable alternative to the current antibiotic-based treatments. In a more recent parallel development, the inorganic mimics of natural enzymes (NanoZymes) have received intensive attention with a promise to catalyze biologically-relevant reactions.13-17 This interest in NanoZymes (nanoparticles as artificial enzymes) is due to the low synthesis cost, high ambient stability, amenity to scale up, tunable catalytic properties,18-23 and in certain cases, selective toxicity against bacteria over mammalian systems. Such properties have recently seen the exploration of NanoZymes for a range of applications, with a strong focus on sensing and diagnostics.13-17 Lately, a new direction of research has emerged where the enzyme-like catalytic activity of nanoparticles has been assessed for its potential in bacterial control.24-28 This concept of NanoZyme-mediated antibacterial activity stems from the ability of nanoparticles to catalyze conversion of H2O2 to hydroxyl radicals (•OH), a reactive oxygen species (ROS) which may then

ACS Paragon Plus Environment

3

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

participate in antibacterial activity.24-28 However, one of the major limitations of the current approach is that most of the known NanoZymes do not depend on an external ‘trigger’ to allow tunability of their antibacterial activity. This means that similar to an antibiotic, these NanoZymes are likely to remain continuously active and may therefore allow opportunistic bacteria to develop resistance. In contrast, regulating the antimicrobial activity of NanoZymes on-demand by using an external ‘trigger’ mechanism may be highly desirable. The use of light as an external trigger to control biological activity is highly attractive as it offers high spatiotemporal resolution without the need for a physical contact.29 In fact, the reversible trans-cis photoisomerization ability of photochromic molecules such as azobenzene and its derivatives under UV-visible photoillumination has been widely explored.30 For instance, we recently showed that the bacterial and biofilm forming ability of carbohydrate-based surfactants containing an azobenzene moiety can be finely controlled using light as an external trigger.31-32 Similar to photochromic molecules, the light-absorbing properties of nanomaterials have also created distinct opportunities in exploiting light to activate nanomaterials for a myriad of processes ranging from photocatalytic splitting of water to photodegradation of environmental pollutants, photodynamic therapies and control of biological processes.30, 33-39 Overall, while light offers remarkable potential as an external trigger to control the antibacterial property, combining the use of light as ‘trigger’ with the NanoZyme activity of nanomaterials remains a relatively unexplored area of research. In the current manuscript, we show the ability of visible light to act as an external trigger for controlling the antibacterial property of semiconducting nanorods (NRs) of CuO. Our investigations begin with developing a new synthesis approach that employs a highly basic tertiary amine (triethylamine, pKa: 10.75) to produce visible light-active CuO NRs. Our further

ACS Paragon Plus Environment

4

Page 5 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

investigations reveal that visible light illumination of CuO NRs modulates their affinity to H2O2, thereby providing a facile control over its enzyme-like catalytic and antibacterial performance. In comparison to non-illuminated conditions, the apparent binding affinity of CuO NRs to H2O2 is observed to increase by over four times (4.35 times) under visible light illumination. This leads to 20 times improvement in the rate of ROS production. These factors remarkably improve the antibacterial performance of photoilluminated CuO NRs by enhancing the •OH radical formation even at low H2O2 concentrations. As such, the salient features of the current study include (i) a new synthesis protocol to fabricate high surface area NanoZymes; (ii) using light as an external ‘trigger’ to catalyze the antibacterial performance; and (iii) a reversible regulation of the NanoZyme catalytic activity by controlling the affinity of the NanoZyme to H2O2. The outcomes of this study are likely to broaden the scope of photoactive NanoZymes for catalyzing antibacterial and other biologically relevant properties. RESULTS AND DISCUSSION A solution based facile approach was developed to synthesize CuO NRs under ambient conditions. In a typical synthesis, when a short chain alkylamine (triethylamine) is introduced to an aqueous solution of copper chloride, the high basicity of triethylamine (pKa: 10.75) allows it to act as a hydrolyzing agent to spontaneously produce CuO NRs. While long chain alkylamines are regularly used as surfactants for shape-directed growth of various crystals,40 the use of small chain organic amines for such purposes is rather limited.41 We chose triethylamine for aqueous synthesis of CuO NRs due to the amphiphilicity of triethylamine molecules that allows the hydrolysis of copper salts to proceed in aqueous solutions without additional alkali, surfactant or a shape-directing agent. We observed that triethylamine directed the crystal growth resulting in the formation of CuO NRs, a process commonly observed with long chain alkylamines.41 The

ACS Paragon Plus Environment

5

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

subsequent washing of CuO NRs with an acetone-methanol mixture and overnight drying resulted in a greyish-black powder. CuO NRs were thoroughly characterized using transmission electron microscopy (TEM), highresolution TEM (HR-TEM), energy-dispersive X-ray spectroscopy (EDS), atomic force microscopy (AFM), X-ray diffraction (XRD) and Raman spectroscopy (Figure 1). TEM image shows rod-like morphology (Figure 1a) with an average length of ca. 2.2 ± 0.67 µm and an average width of ca. 70.1 ± 14.7 nm. The HR-TEM image (Figure 1b) shows a well-ordered structure with continuous lattice fringes and d-spacing of 1.76 Å, 2.52 Å and 2.86 Å, corresponding to the (020), (002) and (011) planes of the monoclinic symmetry of the CuO crystal. The corresponding fast Fourier transform (FFT) pattern shown in Figure 1c further confirms the high crystallinity of these CuO NRs. The EDS elemental map (Figure 1d) and individual mapping of the Cu Kα edge (Figure 1e) suggests that the NRs are composed of Cu and O (Ni signal arising from the nickel TEM grid). The corresponding EDX spectrum (Figure 1f) shows characteristic energy lines of Cu Lα (0.929 keV) and O Kα (0.524 keV) arising from the CuO NRs. The AFM analysis of the NRs (Figure 1g) further confirms the rod-like morphology of CuO, while the height profile suggests that the NRs are, on an average, approximately 30-35 nm thick (Figure 1h). The Bragg reflections obtained from XRD could be perfectly indexed to the standard monoclinic symmetry of CuO (ICDD 45-0937). Additionally, the strong XRD signature at 35.56° corresponding to the (-111) crystal plane of CuO supports that triethylamine selectively directs the crystal growth to achieve nanorod morphology (Figure 1i). The absence of peaks corresponding to Cu(OH)2 or other Cu species shows the ability of the synthesis process to produce high purity material. The broad peak at 22° in the XRD spectrum is attributed to the SiO2 glass substrate on which the active catalyst was drop-casted for analysis.

ACS Paragon Plus Environment

6

Page 7 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

The confocal Raman spectroscopy of individual NRs further reaffirms the purity of CuO (Figure 1j) through presenting three characteristic vibrational peaks at 280, 330 and 614 cm–1, corresponding to the Ag, B1g and B2g modes, respectively.42 X-ray photoemission spectroscopy (XPS), a highly surface sensitive technique, was also used to confirm the oxidation state of Cu and the likely presence of triethylamine on the surface of CuO NRs (Figure S1, Supporting Information). The Cu 2p core level shows two characteristics 2p3/2 and 2p1/2 splitting components (spin–orbit splitting 19.7 eV) with peaks arising at binding energies of ca. 933.5 and 953.2 eV, respectively, corresponding to the Cu2+ oxidation state.43 In addition, two shake-up satellite peaks were also observed, which are due to the non-adiabatic relaxation of the electron cloud that upon photoionization promotes a valence electron to an unoccupied state. This gives rise to the shake-up transition or extra peaks in the XPS spectrum. The presence of such satellite peaks is a characteristic feature specifically when Cu is present in Cu2+ oxidation state.43 The presence of these peaks further confirms that the material is indeed in a highly pure phase CuO NRs. Furthermore, the absence of peaks in the N 1s core level confirms that the triethylamine used during synthesis does not attach to the surface of the CuO NRs. Traditional metal oxide photocatalysts have large band gaps (such as TiO2 with band gap of 3.2 eV), meaning that these materials predominately absorb the ultraviolet light. However, the mutagenicity and associated biohazards of the ultraviolet radiation limit the practical applicability of these materials for biological applications.44 Typical approaches for lowering band gap of semiconductors include doping strategies or hybridizing traditional metal oxides with metal nanoparticles.44-46 Another viable alternative to allow semiconducting materials to absorb visible light is to use semiconductors with smaller band gap energies. In this regard, CuO, a p-type transition metal oxide, is known to possess interesting physicochemical properties,

ACS Paragon Plus Environment

7

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

including a narrow band gap.47 Considering that the use of light as an external ‘trigger’ is central to the production of •OH radicals, we carefully calculated the optical band gap of CuO NRs using optical spectroscopy, and determined the valence band energy levels using photoelectron spectroscopy in air (PESA, Figure S2, Supporting Information). The CuO NRs showed the valence band energy level at 4.98 eV, along with an associated optical band gap of 1.44 eV.48 This should allow CuO NRs to promote the generation of •OH through enhancing its catalytic properties on photoexcitation with visible light (Figure 2). The inherent capability of CuO NRs to generate •OH radicals would mean that this material would show peroxidase-mimic NanoZyme activity (Step 1). The •OH radicals formed as a result of this NanoZyme activity can then participate in killing bacteria without photoexcitation (Step 2). However, when CuO NRs are exposed to visible light, the appropriate energy band structure of nanostructured CuO will photoexcite the charge carriers to enhance the generation of •OH radicals (Step 3). The large amount of these reactive oxygen species (ROS) will drive higher efficiency of bacterial killing on visible light photostimulation (Step 4). Such properties are likely to have a major impact on developing CuO NR based phototunable antibacterial agents. Considering that the inherent catalytic behaviour of CuO NRs to produce •OH radicals is central to its antibacterial performance, the peroxidase-mimic enzymatic behavior of CuO NRs was first assessed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) – ABTS, a substrate for peroxidase group of enzymes (Figure 3). ABTS is a colorless peroxidase substrate, but in the presence of •OH radicals, it is oxidized to a colored product, thereby acting as a model system to evaluate peroxidase-like activity of enzymes and nanomaterials. It is clear that the CuO NRs can efficiently catalyze the oxidation of ABTS in the presence of H2O2 to produce a characteristic green color product (Figure 3a) with maximum absorbance at ca. 415 nm. In contrast, the

ACS Paragon Plus Environment

8

Page 9 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

reaction does not proceed in the absence of H2O2 or the nanoparticles, confirming that the intrinsic peroxidase-like catalytic activity is a characteristic property of CuO NRs. The peroxidase-mimic NanoZyme activity of the CuO NRs was further validated using other peroxidase substrates including 3,3′,5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD). The CuO NRs could also catalyze the oxidation of these peroxidase substrates, such that the colorless TMB oxidized to form a blue product while the colorless OPD converted to a yellow product (Figure 3b). This suggests that the CuO NRs are not specific for a particular peroxidase substrate, but instead show broad-spectrum activity through generating •OH radicals, an observation akin to other NanoZymes.14-15 Subsequent studies involving different catalyst concentrations, temperature, pH, and time-dependent kinetics were performed using ABTS substrate. The NanoZyme concentration dependent activity of CuO NRs reveals that the activity increases as a function of CuO concentration (Figure 3c), wherein the oxidation proceeds rapidly at high CuO NRs concentrations (25 ppm equivalent of Cu+ ions). The absence of catalytic activity in control experiments employing supernatants isolated from the aqueous dispersions of CuO NRs ascertained that the observed nanozyme activity was not due to the potential leaching of Cu ions during the experiments (Figure S3, Supporting Information). The peroxidase-mimic activity of CuO NRs was also compared with commercially sourced CuO microparticles (MPs). The CuO NRs outperform the catalytic activity observed for CuO MPs as evident from the time-dependent kinetics (Figure S4, Supporting Information). To understand the reason for this higher activity, the surface areas and particle morphology of the synthesized CuO NRs and commercially sourced CuO MPs were determined using Brunauer–Emmett–Teller (BET) analysis and electron microscopy, respectively. The surface area of NRs (44.96 m2 g-1) was found to be over 10-fold higher than that obtained for MPs (4.32 m2 g-1), suggesting that the

ACS Paragon Plus Environment

9

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

surface area plays a major role in enhancing the nanozyme activity of CuO NRs. It is also notable that the surface area of CuO NRs prepared in this study is higher than those observed for other CuO materials reported in the literature (Table S1, Supporting Information). Additionally, while the CuO synthesized using our approach yields a rod-like morphology, commercial CuO showed highly irregular shape and size (Figure S5, Supporting Information). On an average, the particle size was considered sub-10 µm as suggested from the commercial source (CuO particles from Sigma Aldrich – 208841). The differences in the morphological characteristics between CuO NRs and CuO MPs correlate well with the obtained surface area, which was ~10-folds higher for CuO NRs. This suggests that in addition to the surface area, the potential influence of morphology in the enhanced NanoZyme activity of the NRs cannot be negated. Further, the peroxidase-like activity of CuO NRs was also found to be dependent on the reaction pH, temperature and time (Figure S6, Supporting Information). For instance, the CuO NRs show nanozyme activity over a pH range of 3–6 with maximum activity at pH 4. The CuO NRs also show catalytic activity over broad temperature range of 25–60 °C with maximum activity at 45 °C. The catalytic activity increases linearly as a function of reaction time, with the activity saturating to the maximum value within 20 minutes at the CuO concentration of 25 ppm. Conversely, at lower CuO concentrations, no saturation point is observed, suggesting that the catalyst concentration plays a vital role in promoting the reaction rapidly. For biomedical applications, it is essential that the NRs have high catalytic activity and stability at physiological pH (6–7.4) and temperature (37 ºC).49 Based on these results, it is clear that CuO NRs show excellent catalytic activity and stability at physiological conditions (~60% of the activity is retained at pH 6, and ~80% of the activity is retained at 37 ºC).

ACS Paragon Plus Environment

10

Page 11 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

Having established the intrinsic NanoZyme characteristics of CuO NRs, the next step was to determine whether visible light could be used a trigger to enhance this catalytic performance. As evident from Figure 2, the favorable energy band structure of CuO NRs should, in principle, allow their stimulation by visible light (λ≥400 nm). Similar to the enhancement in the photocatalytic performance observed in traditional metal nanoparticles such as Au,9 Ag33 and Cu (surface plasmon resonance effect) and traditional semiconducting materials such as TiO239 and ZnO50, the catalytic activity of CuO NRs showed significant catalytic enhancement under visible light irradiation (Figure S7, Supporting Information). A time-dependent activity profile showed that under visible light irradiation, CuO NRs promoted rapid oxidation of ABTS that reached a plateau within 10 min, in contrast to 20 min when the same reaction proceeds under nonphotoirradiated conditions. To understand the mechanism of this NanoZyme activity, we investigated the relative •OH radical generation ability of CuO NRs in the presence and absence of visible light by employing terephthalic acid as a fluorogenic probe. On reaction with •OH radicals, the non-fluorescent terephthalic acid converts to 2-hydroxy terephthalic acid with fluorescence at ca. 435 nm.51 CuO NRs in the presence of H2O2 show a fluorescence response, confirming their ability to produce •

OH radicals (Figure S8, Supporting Information). Further, this response increases by ~2.4 times

when the same reaction is carried out in the presence of visible light. This suggests that when the reaction proceeds in the presence of visible light, the catalytic activity gets modulated either through increased affinity of the CuO NRs towards the ABTS substrate and/or to H2O2 leading to enhanced •OH radical generation. We also investigated the relative •OH radical generation ability of CuO NRs with increasing concentration of H2O2 in the presence and absence of light illumination conditions. Figure S9 in the Supporting Information clearly demonstrates that a

ACS Paragon Plus Environment

11

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

significant amount of •OH radical is generated even at low 1 mM H2O2 concentration. In fact, the •

OH radicals production is significantly enhanced when the reaction is carried out under light

illumination conditions. The mechanism of H2O2 degradation in typical transition metal oxides has been previously evaluated and it is believed that H2O2 molecules adsorb on the surface of the nanoparticles and the nanoparticle catalyst, the nanoparticles then break up the O-O bond of H2O2 to generate two •OH radicals.21 It is expected that a similar mechanism might be involved during the CuO NR-mediated decomposition of H2O2 to generate •OH radicals. Further, different reports have outlined the generation of different ROS including singlet oxygen radical, superoxide radical, or •OH radical when using CuO particles.52-55 This suggests that the generation of ROS when using CuO as a catalyst may occur through different mechanisms. To study the potential role of other ROS in the NanoZyme activity of CuO NRs synthesized ion our study, we performed additional experiments using specific dyes that can capture the targeted ROS. Our experiments suggest that the CuO NRs do not produce either singlet oxygen or superoxide radical species (Figure S10, Supporting Information), and produce only •OH radicals. In a recent study by Martinez et al., it was suggested that •OH radical is the main ROS generated by CuO nanoparticles in a H2O2 catalyzed reaction.52 Based on this and the TA-based quenching experiments, we can conclude that •OH radical is the predominant ROS responsible for the NanoZyme activity of CuO NRs. To understand the mechanism of this photostimulated increase in •OH radical production, the kinetic parameters of the reactions involving ABTS and H2O2 were determined22 by calculating the Michaelis-Menten constant (Km) and the maximum initial velocity (Vmax) of the reaction in the absence and presence of visible light. Typical Michaelis-Menten curves were observed for both ABTS and H2O2 (Figure S11, Supporting Information) irrespective of whether the reaction

ACS Paragon Plus Environment

12

Page 13 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

proceeds with and without visible light stimulation. The double reciprocal Lineweaver-Burk plots of the activity of CuO NRs showed characteristic linear fits (Figure 4) suitable for obtaining the Km and Vmax values (Table 1). Given that the Km represents the affinity of the NanoZyme to the substrate, a lower Km value would mean higher affinity of CuO NRs to the substrates. Under visible light irradiation, the calculated Km values for both ABTS and H2O2 were lower than those observed without photoillumination. In particular, the Km for H2O2 significantly reduced from 14.8 mM to 3.4 mM, a decrease of 4.35 folds; while the Km for ABTS showed a marginal decrease of 1.5 fold under visible light irradiation. This suggests that under photoirradiated conditions, the affinity of CuO NRs towards H2O2 remarkably improves, implying that fewer H2O2 molecules will be required to produce higher amounts of •OH radicals. On the other hand, the Vmax for both ABTS and H2O2 consumption were higher during photoillumination in comparison to the dark conditions, with 2.7 and 19.8 fold improvements, respectively. The higher Vmax values under photoirradiated conditions imply that the rate of production of •OH radicals will be over ~20 times faster than under non-irradiated conditions. These investigations provide clear evidence that visible light acts as an outstanding trigger to significantly enhance the oxidative properties of CuO NRs through producing larger quantities of •

OH radicals (low Km) at a faster rate (high Vmax).

The ability to controllably tune the affinity of CuO NRs to H2O2 and thereby influencing the •OH radical production using light as an external ‘trigger’ offers a powerful strategy to employ these materials as antibacterial agents. Since the highly oxidizing •OH radicals are expected to participate in killing bacteria, to validate this concept, we employed Escherichia coli (E. coli) as a model Gram negative bacterium and studied the antibacterial profile of CuO NRs under different conditions. The CuO NRs in the absence of H2O2 showed concentration-dependent

ACS Paragon Plus Environment

13

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

antibacterial activity; however, they were found ineffective in killing bacteria within 2 h with lower than 20 ppm of the catalyst concentrations (Figure S12, Supporting Information). At these concentrations, irrespective of the pH (6.0 or 7.0) or the absence/presence of visible light as trigger, CuO NRs could cause only less than 20% bacterial cell death. Similarly, 1 mM of H2O2 alone in the absence of CuO NRs had minimal influence (less than 10% cell death) on the bacterial viability (Figure S13, Supporting Information). This basal antibacterial activity of H2O2 was also found independent of the pH of the bacterial growth medium or the photoillumination conditions. In the next stage, the ability of CuO NRs to act as a NanoZyme in the presence of H2O2, and thereby driving the generation of •OH radicals to enhance bactericidal activity was evaluated (Figure 5). For these studies, it was important that the chosen concentrations of CuO NRs and H2O2 were such that these individual components remain independently non-toxic to bacteria (less than 20 ppm of CuO NRs and less than 1 mM of H2O2). In comparison with the control groups (CuO NRs in the absence of H2O2 illuminated with visible light), the CuO NRs supplemented with H2O2 caused significant bactericidal effect within 2 h of exposure. The enhancement of this antibacterial activity of CuO NRs in the presence of H2O2 was dependent on the concentration of CuO NRs, the pH of the bacterial media and the photo-illumination conditions. For instance, while 20 ppm of CuO NRs in the absence of H2O2 but in the presence of light caused only ~10% cell death; the bacterial toxicity in the presence of H2O2, even under dark conditions increased to ~27% at pH 7.0, which further increased to ~48% at pH 6.0. The superior antibacterial performance of CuO NRs at pH 6.0 corroborates well with the pHdependent enzyme-mimic studies, where pH 6.0 showed better NanoZyme activity over pH 7.0. Under similar conditions as above at pH 6.0, but with visible light illumination, the suppression

ACS Paragon Plus Environment

14

Page 15 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

of bacterial growth further enhanced remarkably to ~92%. This establishes the ability of light to act as a trigger to enhance the antibacterial activity of CuO NRs through generating •OH radicals. A time-dependent study involving bacterial exposure to CuO NRs and H2O2 for different durations of photoillumination further emphasizes the direct role of light as a trigger in enhancing antibacterial performance (Figure S14, Supporting Information). At initial time points (30 min), the antibacterial performance between non-illuminated and illuminated conditions is rather similar. However, as the photo-exposure time increases, the difference in the antibacterial activity profile becomes remarkably larger to an extent that photoillumination of CuO NRs leads to killing of more than twice the number of bacteria than under standard conditions. In addition, the relative increase in the •OH radical production at 1 mM H2O2 under dark and light illumination conditions (~2.8 times improvement) correlates rather well with the antimicrobial profile of CuO NRs in the presence of 1 mM H2O2 (~2.5 times improvement from dark to light) as shown in Figure 5. It is important to note that microorganisms such as E. coli also produce H2O2 in situ during their growth under aerobic conditions.56 This means that an effectively higher H2O2 concentration will be available to bacteria during their exposure to CuO NRs in the presence of externally-added 1 mM H2O2. These experiments clearly demonstrate the significant impact of highly active CuO NR NanoZymes in acting as an outstanding antimicrobial agent even in the presence of small amounts of H2O2. To further validate that the antibacterial performance is indeed due to the generation of •OH radicals and not potentially due to other factors, a reactive oxygen species suppression study was performed using ascorbic acid (AA), a well-known antioxidant.57 To establish the optimum concentration of AA, three independent concentrations of AA (0.01, 0.1 and 0.5 mM) were first assessed for their ability to quench the peroxidase-like NanoZyme activity of CuO NRs. The

ACS Paragon Plus Environment

15

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

quenching of the catalytic activity was monitored by recording the absorbance of the ABTS oxidation product over time under dark and visible light illumination. As evident from Figure S15, Supporting Information, the rate of ABTS oxidation reduced with increasing concentrations of AA, such that the maximum quenching of •OH radicals was observed at 0.5 mM concentration. This concentration of AA was then combined with different concentrations of CuO NRs to study its effect on bactericidal activity against E. coli (Figure S16, Supporting Information). In the presence of AA, the CuO NRs almost completely lose their ability to kill E. coli under both dark and visible light illumination conditions. This is primarily because the AA molecules sequester any reactive oxygen species that are generated by CuO NRs, thereby avoiding bacterial toxicity. Considering that reactive oxygen species typically result in the rupture of bacterial cell surface, the influence of ROS generated by the CuO NRs on the morphology of E. coli was further investigated using TEM (Figure 6). The untreated E. coli cells show a typical rod-shaped morphology with intact cell walls and a well-preserved ultrastructure (Figure 6a). The exposure of bacteria to CuO NRs in the presence of H2O2 leads to loss of their morphological integrity by becoming rough and wrinkled. While this effect is evident only on few cells under non-irradiated conditions (Figure 6b), a significantly larger proportion of bacteria are seen to lose their cellular integrity under photoexcitation conditions (Figure 6c, and additional images in Figure S17, Supporting Information). Further, the damaging effect of ROS species under visible light photoexcitation enhances to the level that it causes noticeable physical damage to the bacterial cells (highlighted by arrows). This observation may be explained based on the basis that under normal scenario, the bacterial cells are equipped with endogenous antioxidants such as superoxide dismutase and catalase enzymes that have the ability to neutralize H2O2 and ROS

ACS Paragon Plus Environment

16

Page 17 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

species to reduce the cellular stress.58 However, the outstanding ability of CuO NRs to produce •

OH radicals, particularly in the presence of visible light allows excessive ROS production

within a short time frame (Vmax under photoexcitation conditions was found to be ~20 times higher than non-irradiated conditions), leading to rapid oxidative degeneration of bacteria.

CONCLUSION In summary, we have developed a simple yet versatile strategy that allows light as an external ‘trigger’ to control the NanoZyme-catalyzed antibacterial activity of CuO nanorods. Photoillumination modulates the peroxidase-mimic catalytic performance of CuO nanorods through enhancing their affinity to H2O2. Additionally, the favorable energy band structure of CuO nanorods remarkably accelerates the ROS production by 20 times under visible light, even at relatively low concentrations of H2O2. This leads to improved antibacterial performance against Gram negative E. coli through rapid denaturation of bacterial cells via ROS-induced physical damage. This work paves a way forward to control the biocatalytic activity of NanoZymes using light as a modular switch. EXPERIMENTAL Materials and Reagents: Cupric chloride (CuCl2.2H2O) was purchased from BDH chemicals Ltd., 3,3′,5,5′-Tetramethylbenzidine (TMB), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD), triethylamine (TEA), L-ascorbic acid (AA), terephthalic acid (TA) 9,10-anthracenediyl-bis (methylene) dimalonic acid (ABMDMA), and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Australia. Ethanol (C2H5OH, 100%) and hydrogen

ACS Paragon Plus Environment

17

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

peroxide (H2O2, 30% w/w) were purchased from Chem-Supply Pty Ltd. Amyl media nutrient broth was purchased from Amyl Media Pty Ltd. Sodium acetate trihydrate (anhydrous) and disodium hydrogen phosphate were purchased from BDH Chemicals Ltd. Escherichia coli (E. coli ATCC 25922) bacterial strain was purchased from Southern Biologicals. Milli-Q water (18.2 MΩ cm) was obtained from Milli pore water purification system and was used for the preparation of all solutions. Synthesis of CuO nanostructures: A solution-based approach was used to obtain CuO NRs. In a typical synthesis, 0.2 M copper chloride (II) dihydrate (CuCl2.2H2O) was first dissolved in 40 mL of deionised (MilliQ) water in a round bottom flask. This mixture was heated to 80 °C and was maintained at this temperature under stirring (500 rpm) conditions. To this mixture, 5 mL of triethylamine was added and stirred continuously for one hour after which the solution was allowed to cool at room temperature. The precipitate obtained from this reaction was washed in a acetone: methanol (1:1 v/v) mixture and dried in vacuum to obtain a greyish-black powder. Materials characterization: Samples for transmission electron microscopy (TEM) and highresolution TEM (HR-TEM) were prepared by drop coating the samples on a holey carbon TEM grid and holey carbon-coated nickel grid, respectively. The samples were analysed using JEOL 1010 and JEOL 2200FS TEM instruments, operated at an accelerating voltage of 100 kV and 200 kV, respectively. EDX maps and spectra were collected using a JEOL 2200FS TEM instrument in STEM bright field mode with a Bruker detector. The scanning electron microscopy (SEM) images of the commercial CuO MPs and CuO NRs were obtained using FEI Quanta SEM instrument operated at an accelerating voltage of 10 kV. X-Ray Diffraction (XRD) was obtained using Bruker AXS D4 Endeavour – Wide Angle XRD with Cu Kα radiation (λ: 1.5406 Å). X-ray photoemission spectroscopy (XPS) measurements were carried out using Thermo K-Alpha XPS

ACS Paragon Plus Environment

18

Page 19 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

instrument. The Cu 2p, O 1s and N 1s core level spectra were recorded with unmonochromatized Al Kα radiation (photon energy of 1486.7 eV) at a pass energy of 20 eV and an electron take-off angle of 90°. The overall resolution was 0.1 eV for all XPS measurements. The core level spectra were background corrected using the Shirley algorithm and chemically distinct species were resolved using nonlinear least squares fitting procedure.59 The binding energies (BEs) were aligned with an adventitious carbon binding energy of 285 eV. The Cu ions were quantified using Agilent 4200 Microwave Plasma-Atomic Emission Spectrometer (MPAES). Raman spectrum was obtained using a Horiba LabRAM HR Evolution micro-Raman system equipped with 532 nm laser (0.5 µm lateral resolution, 0.25 s exposure) using a 100 × objective. Raman spectra was background corrected using an in-house developed smoothing free algorithm.60 Atomic-force microscopy (AFM) images were obtained using a Bruker Dimension Icon instrument in tapping mode. The absorbance spectra were obtained using Envision multimode plate reader (Perkin Elmer). Fluorescence spectrometry was performed using Fluromax-4 spectrofluorometer at an excitation wavelength of 315 nm. The specific surface area of the CuO NRs was measured using nitrogen (N2) adsorption isotherms at –196 °C using a Braunauer-Emmett- Teller (BET) analysis method on a Micromeritics (ASAP 2000) instrument. For all light illumination experiments, a broadband white 12 W light emitting diode (LED) lamp (Techbrands by Electus Distribution Pty Ltd. New South Wales, Australia) was used where the distance between the lamp and the solution was ~5.6 cm with an intensity of 15.61 mW cm-2. Optical absorption spectra of CuO NRs suspended in water were conducted on an Agilent Cary 7000 spectrophotometer equipped with an integrating sphere by positioning the sample inside the sphere to eliminate the scattering contribution. Tauc analysis was conducted assuming indirect

ACS Paragon Plus Environment

19

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

band gap transition. Photoelectron Spectroscopy in Air (PESA) was conducted on a Riken Keiki AC-2 spectrometer using 20 nW radiation on samples deposited on glass substrates. Peroxidase-like catalytic performance of CuO nanostructures: The peroxidase-like nanozyme activity of CuO NRs was investigated through its ability to oxidize peroxidase substrate (ABTS, TMB or OPD) to colored product (green, blue, yellow) in the presence of H2O2 [25 ppm CuO, 0.1 mM ABTS/ TMB/ OPD and 100 mM H2O2 in 50 mM sodium acetate buffer (pH 4) at 37 °C after 20 min of reaction]. These colored products were characterized by a typical absorbance signal at ca. 415, 650 and 450 nm, respectively. Further, using ABTS as the peroxidase substrate, (i) the CuO NR concentration-dependent activity (0-25 ppm equivalent of Cu ions), (ii) pHdependent activity (pH 3–12 buffers), and (iii) temperature-dependent activity (10–60 °C) in pH 4 sodium acetate buffer, were determined. For concentration-dependent studies, the absorbance of oxidized ABTS at 415 nm was plotted while defining the highest absorbance value as 100% relative activity. Steady state kinetic analysis was performed at a fixed Cu ion concentration (25 ppm) by varying the substrate concentration from 0.01 – 0.3 mM for ABTS and 1 – 1000 mM for H2O2. The obtained data were fitted to Michaelis-Menten and Lineweaver-Burk plots using OriginPro 2016 and the apparent enzyme kinetic parameters were calculated. Mechanism of peroxidase-like activity: The generation of •OH radicals by the CuO NRs in the presence and absence of light illumination was determined using a fluorescence assay. This typically involved the use of terephthalic acid (TA) as a probe molecule, which reacts with •OH radicals to form highly fluorescent 2-hydroxyterephthalic acid.51 The experiment was carried out by mixing 10 mM H2O2, 0.5 mM terephthalic acid and 25 ppm of CuO NRs in 1 mL of 50 mM sodium acetate buffer (pH 4). The fluorescence intensity was measured using an excitation wavelength of 315 nm. Singlet oxygen generation was monitored by the potential photo-

ACS Paragon Plus Environment

20

Page 21 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

bleaching of ABMDMA dye in presence of CuO NRs and H2O2 under dark and light illumination condition. ABMDMA can be potentially photobleached by singlet oxygen to form its corresponding endoperoxide, which will be reflected by the decrease in the absorbance at ca. 400 nm. The experiment was performed by mixing 0.25 mM ABMDMA, 1 mM H2O2 and 20 ppm of CuO NRs in 50 mM sodium acetate buffer (pH 6). Control experiments were performed without adding CuO NRs and H2O2 in the reaction medium. Superoxide radical generation was monitored using an MTT assay. MTT is a tetrazolium dye that can be reduced by superoxide radicals to give a colored formazan product with absorbance maxima at ca. 570 nm. The assay was performed by mixing 0.1 mM MTT, 1 mM H2O2 and 20 ppm of CuO NRs in 50 mM sodium acetate buffer (pH 6). Control experiments were performed without adding CuO NRs and H2O2 in the reaction medium. Antimicrobial experiments: A single freshly revived colony of an E. coli pure culture was subcultured in a shaking incubator in 10 mL amyl nutrient media (13 g powder in 1 L of Milli-Q water) at 37 °C, 100 rpm overnight. The cell density of the overnight grown bacteria was adjusted to 0.5 OD by recording optical density at 600 nm (OD600) using a UV-vis spectrophotometer. A measured volume of 1 mL was transferred to 100 mL of fresh media for a further conditioning at 37 °C, 250 rpm for 3 h. Finally, bacterial cell density was adjusted to 0.1 OD containing approximately 1 x 108 CFU mL-1 (CFU: colony forming unit) for the antibacterial experiments. The antibacterial activity of the CuO NRs towards E. coli was determined by recording the OD600 using microplate reader in absence and presence of H2O2. The minimum cytotoxicity of nanoparticles and H2O2 to E. coli was first determined by varying CuO nanoparticle (0 to 100 ppm) and H2O2 (0 to 100 mM) concentrations, independently at pH 6.0 and 7.0 under both dark and light illumination conditions. The antibacterial activity of CuO NRs

ACS Paragon Plus Environment

21

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 37

against E. coli was determined at different pH (6, and 7) using 5, 10 and 20 ppm of CuO NRs in absence and presence of 1 mM H2O2 with a total reaction volume of 1 mL in 24-well plates incubated for 2 h. The pH values of nutrient medium were adjusted using hydrochloric acid. In the control group, equal amount of sterilized Milli-Q water and 5, 10 and 20 ppm of CuO NRs or H2O2 (1 mM) alone were added to the solution of E. coli under the same conditions. The relative cell death was calculated according to the following equation: Cell death (%) = 100 - [((TOD600 nm /COD600 nm) × 100)] where COD600 nm is the optical density of the control, and TOD600 nm is the optical density of the treatments with CuO NRs with H2O2 or CuO NRs or H2O2 alone. Preparation of TEM samples for bacterial studies: After treatment with both CuO NRs and H2O2, the CuO NRs were removed from the bacterial solution by centrifugation at 500 rpm. Then, bacterial pellet was collected in PCR tubes and washed with 0.2 M cacodylate buffer and fixed in 2% paraformaldehyde (ProSciTech, Australia) + 2.5% glutaraldehyde (ProSciTech, Australia) in 100 mM cacodylate buffer (pH 7.4) for 2 hrs. After fixation, the samples were rinsed thrice in 100 mM cacodylate buffer. The samples were post-fixed in 1% osmium tetroxide (ProSciTech, Australia) in distilled water for 1.5 hours at room temperature. After fixation, the samples were washed thrice in distilled water for 10 mins. The samples were dehydrated in graded series of ethanol (from 50 to 100%) for 10 minutes for each step and were dehydrated in absolute acetone for 30 mins. After extensive dehydration, the samples were infiltrated with different ratios of acetone and Spurrs resin with increasing resin concentration overnight. The samples were finally infiltrated in fresh Spurs resin twice for 2 hours each. The resin infiltrated samples were polymerised in the oven at 70 °C. Ultra-thin sections of 90 nm thickness was cut,

ACS Paragon Plus Environment

22

Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

post-stained and imaged using a JEOL 1010 microscope operated at an accelerating voltage of 80 kV. Effect of quencher on the antimicrobial performance of CuO NRs: CuO NRs were mixed with three concentrations of ascorbic acid (0.01, 0.1 and 0.5 mM) and peroxidase-like activity was evaluated as described above by recording the changes in absorbance at 415 nm for 20 min under both dark and light illumination conditions. Similarly, bacterial suppression studies were also performed in presence of CuO NRs concentrations (5, 10 and 20 ppm) and 1 mM H2O2 at pH 6 by adding 0.5 mM ascorbic acid in the reaction medium under both dark and light illumination conditions. Control experiments were performed without adding CuO NRs in both of the reaction medium.

ACS Paragon Plus Environment

23

ACS Applied Nano Materials

a

b

c

2.86 Å (011)

2.52 Å (002)

1.76 Å (020)

100 nm

5 1/nm

f

Intensity [a.u.]

e

d

Ni Cu

Cu O C

0

Cu

2

4

6

8

10

Energy [KeV]

h

35

Intensity [a.u.]

25 20 15 10

Cu O

B1g B2g

5

1 400 nm

0 0

0

j A1g

i

30

Intensity [a.u.]

g Height [nm]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

nm

100

125

250

375

Distance [nm]

20

40

60

80

2 [degree]

270

405

540

675

Wavenumber [cm-1]

Figure 1. Characterization of CuO NRs. (a) TEM, (b) HR-TEM, (c) FFT pattern, (d-e) EDX maps, (f) EDX spectrum, (g) AFM image, (h) AFM height profile measured at marker 1 indicated in (g), (i) XRD pattern with atomistic model of CuO, and (j) confocal Raman spectrum from a single NR.

ACS Paragon Plus Environment

24

Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

2 1 Intrinsic ability of CuO Nanozyme to produce · OH radicals H 2 O2

-3

radicals kill bacteria

CB 3.54

-4 OH·

H 2 O2

H 2 O2

· OH

-5

VB 4.98 3 Visible light ‘triggers’

eV

CuO and enhances the production of · OH - 3 radicals

H2O2 H 2 O2

-4

CB 3.54

4 High amounts of ·OH

kill more bacteria

OH· OH·

H 2 O2 OH·

-5

VB 4.98

Figure 2. Mechanism of NanoZyme-catalysed antibacterial performance of CuO NRs. The favourable energy band structure of CuO NRs allows it to spontaneously produce •OH radicals (Step 1), which in turn makes CuO NRs a good antibacterial agent (Step 2). A narrow bang gap further allows visible light to be used as an external ‘trigger’ to catalyse CuO NR-mediated enhanced production of •OH radicals (Step 3). This enhanced light-mediated •OH radical production kills bacteria more efficiently (Step 4).

ACS Paragon Plus Environment

25

ACS Applied Nano Materials

(b) (ii)

(iii)

(iii) (ii) (i)

(c) (i)

Absorbance [a.u.]

(i)

(ii) (iii)

(iii) (ii) (i)

Peroxidase activity [%]

(a) Absorbance [a.u.]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 37

100 80 60 40 20 0

400 500 600 700

400 500 600 700

Wavelength [nm]

Wavelength [nm]

0

5 10 15 20 25

CuO NRs conc. [ppm]

Figure 3. Peroxidase-mimic activity of CuO NRs. (a) UV-visible absorbance spectra of ABTS oxidation under different conditions: (i) ABTS + H2O2, (ii) CuO NRs + ABTS, (iii) CuO NRs + ABTS + H2O2. (b) UV-visible absorbance spectra of different peroxidase substrates using CuO NRs in the presence of H2O2: (i) OPD, (ii) TMB, and (iii) ABTS. Insets show the color of postreaction solutions. (c) Concentration dependent NanoZyme activity of CuO NRs against ABTS substrate.

ACS Paragon Plus Environment

26

Page 27 of 37

(a)

(b)

Y = 1.53 E6 X + 2.44 E7 1.8 R2 = 0.99

1/Velocity [108 M-1s]

1/Velocity [108 M-1s]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

1.2 Y = 3.31 E5 X + 9.05 E6 R2 = 0.99

0.6

Y = 2.67 E9 X + 1.81 E8 2 30.0 R = 0.97

20.0

10.0

Y = 3.11 E7 X + 9.20 E6 R2 = 0.98

0.0 0.0 0

20 40 60 80 100 -1

1/ABTS conc. [mM ]

0.0

0.3

0.6

0.9

1/H2O2 conc. [mM-1]

Figure 4. Steady state kinetics of peroxidase-mimic CuO NRs. Double reciprocal Lineweaver-Burk plots of the catalytic activity of CuO NRs at a fixed concentration of one substrate versus varying concentration of the second substrate for (a) ABTS and (b) H2O2. Blue and red fittings indicate the experiments performed in the absence and presence of visible light illumination, respectively.

ACS Paragon Plus Environment

27

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

Figure 5. Light triggered antibacterial performance of CuO NRs. Comparison of the CuO NanoZyme-catalysed antibacterial activity under dark and photoexcited conditions at (a) pH 6.0 and (b) 7.0, respectively, as represented from % cell death of E. coli incubated for 2 h with different concentrations of CuO NRs and a fixed concentration of 1 mM H2O2. Bacteria treated with different concentrations of CuO NRs in the absence of H2O2, but under visible light illuminations served as the control group.

ACS Paragon Plus Environment

28

Page 29 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

Figure 6. Physical damage to bacterial cells through ROS-mediated antibacterial activity. Cross-section TEM images of E. coli under different conditions: (a) untreated, (b) CuO NRs + H2O2 without irradiation, and (c) CuO NRs + H2O2 with visible light irradiation

ACS Paragon Plus Environment

29

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

Table 1. Comparison of the kinetic parameters of CuO NRs driven reactions under nonilluminated conditions and under visible light illumination. ABTS

H2O2

Conditions K / Vmax / 10- Km / Vmax / 10-8 m 8 mM mM Ms-1 Ms-1 Dark

0.06

4.1

14.8

0.6

Light

0.04

11.1

3.4

10.9

ACS Paragon Plus Environment

30

Page 31 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

ASSOCIATED CONTENT Supporting Information. Additional materials characterization including XPS, optical band gap measurement and calculation, SEM and TEM images of CuO and E. coli cells, BET surface area analysis; NanoZyme assay (time-, pH- and temperature-dependent studies); ROS generation studies (TA, ABMDMA and MTT-assays); steady state kinetic analysis; additional antibacterial assays including CuO concentration- and time-dependent experiments as well as antibacterial performance in the presence of ascorbic acid as a quencher; and a table comparing the BET surface area of CuO-based nanomaterials reported in the literature.

AUTHOR INFORMATION Corresponding Author * [email protected]; [email protected]; [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources Australian Research Council (ARC) Future Fellowship (FT140101285); ARC Discovery (DP170103477) grant; The Commonwealth of Australia Endeavour Research Fellowship; Ian Potter Foundation; The Commonwealth of Australia for an Australian Postgraduate Award; RMIT University for VC postgraduate Fellowship and Vice Chancellor Fellowship.

ACS Paragon Plus Environment

31

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 37

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT N. Karim thanks RMIT University for a VC postgraduate fellowship. M. Singh and P. Weerathunge thank the Commonwealth of Australia for Australian Postgraduate Awards. V. Bansal thanks the Australian Research Council (ARC) for a Future Fellowship (FT140101285). R. Ramanathan, S. Walia and E. Della Gaspera acknowledge RMIT University for Vice Chancellor Fellowships. V. Bansal and R. Ramanathan acknowledge the Australian Research Council (ARC) for research support though an ARC Discovery (DP170103477) grant. S. Singh thanks the Commonwealth of Australia for an Endeavour Research Fellowship. The authors recognize the generous support of the Ian Potter Foundation towards establishing an Ian Potter NanoBioSensing Facility at RMIT University. Authors acknowledge the support from the RMIT Micro Nano Research Facility (MNRF) and RMIT Microscopy and Microanalysis Facility (RMMF) for technical assistance and providing access to characterization facilities. The authors would like to thank CSIRO for providing access to PESA. REFERENCES 1.

2. 3. 4. 5.

Rizzello, L.; Pompa, P. P., Nanosilver-Based Antibacterial Drugs and Devices: Mechanisms, Methodological Drawbacks, and Guidelines. Chem. Soc. Rev. 2014, 43, 1501-1518. Hawkey, P. M., The Growing Burden of Antimicrobial Resistance. J. Antimicrob. Chemother. 2008, 62, i1-i9. Muñoz-Bonilla, A.; Fernández-García, M., Polymeric Materials with Antimicrobial Activity. Prog. Polym. Sci. 2012, 37, 281-339. Chernousova, S.; Epple, M., Silver as Antibacterial Agent: Ion, Nanoparticle, and Metal. Angew. Chem. Int. Ed. 2013, 52, 1636-1653. von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Häbich, D., Antibacterial Natural Products in Medicinal Chemistry—Exodus or Revival? Angew. Chem. Int. Ed. 2006, 45, 5072-5129.

ACS Paragon Plus Environment

32

Page 33 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

6.

7.

8.

9.

10.

11.

12.

13.

14. 15. 16.

17.

18.

19.

Buffet-Bataillon, S.; Tattevin, P.; Bonnaure-Mallet, M.; Jolivet-Gougeon, A., Emergence of Resistance to Antibacterial Agents: The Role of Quaternary Ammonium Compounds — A Critical Review. Int. J. Antimicrob. Agents 2012, 39, 381-389. Hajipour, M. J.; Fromm, K. M.; Akbar Ashkarran, A.; Jimenez de Aberasturi, D.; Larramendi, I. R. d.; Rojo, T.; Serpooshan, V.; Parak, W. J.; Mahmoudi, M., Antibacterial Properties of Nanoparticles. Trends Biotechnol. 2012, 30, 499-511. Daima, H. K.; Selvakannan, P. R.; Shukla, R.; Bhargava, S. K.; Bansal, V., Fine-Tuning the Antimicrobial Profile of Biocompatible Gold Nanoparticles by Sequential Surface Functionalization Using Polyoxometalates and Lysine. PLoS ONE 2013, 8, e79676. Carnovale, C.; Bryant, G.; Shukla, R.; Bansal, V., Size, Shape and Surface Chemistry of Nano-Gold Dictate its Cellular Interactions, Uptake and Toxicity. Prog. Mater. Sci. 2016, 83, 152-190. Berean, K. J.; Adetutu, E. M.; Ou, J. Z.; Nour, M.; Nguyen, E. P.; Paull, D.; Mcleod, J.; Ramanathan, R.; Bansal, V.; Latham, K., A Unique in vivo Approach for Investigating Antimicrobial Materials Utilizing Fistulated Animals. Sci. Rep. 2015, 5, Article no. 11515. Azam, A.; Ahmed, A. S.; Oves, M.; Khan, M. S.; Habib, S. S.; Memic, A., Antimicrobial Activity of Metal Oxide Nanoparticles against Gram-Positive and Gram-NSegative Bacteria: A Comparative Study. Int. J. Nanomedicine 2012, 7, 6003-6009. Davoudi, Z. M.; Kandjani, A. E.; Bhatt, A. I.; Kyratzis, I. L.; O'Mullane, A. P.; Bansal, V., Hybrid Antibacterial Fabrics with Extremely High Aspect Ratio Ag/AgTCNQ Nanowires. Adv. Funct. Mater. 2014, 24, 1047-1053. Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X., Intrinsic Peroxidase-Like Activity of Ferromagnetic NSanoparticles. Nat. Nanotechnol. 2007, 2, 577-583. Wang, X.; Hu, Y.; Wei, H., Nanozymes in Bionanotechnology: From Sensing to Therapeutics and Beyond. Inorg. Chem. Front. 2016, 3, 41-60. Wei, H.; Wang, E., Nanomaterials with Enzyme-Like Characteristics (Nanozymes): Next-Generation Artificial Enzymes. Chem. Soc. Rev. 2013, 42, 6060-6093. Sharma, T. K.; Ramanathan, R.; Weerathunge, P.; Mohammadtaheri, M.; Daima, H. K.; Shukla, R.; Bansal, V., Aptamer-Mediated ‘Turn-Off/Turn-On' Nanozyme Activity of Gold Nanoparticles for Kanamycin Detection. Chem. Commun. 2014, 50, 15856-15859. Weerathunge, P.; Ramanathan, R.; Shukla, R.; Sharma, T. K.; Bansal, V., AptamerControlled Reversible Inhibition of Gold Nanozyme Activity for Pesticide Sensing. Anal. Chem. 2014, 86, 11937-11941. Nagvenkar, A. P.; Gedanken, A., Cu0.89Zn0.11O, A New Peroxidase-Mimicking Nanozyme with High Sensitivity for Glucose and Antioxidant Detection. ACS Appl. Mater. Interf. 2016, 8, 22301-22308. Chen, W.; Chen, J.; Feng, Y.-B.; Hong, L.; Chen, Q.-Y.; Wu, L.-F.; Lin, X.-H.; Xia, X.H., Peroxidase-like Activity of Water-soluble Cupric Oxide Nanoparticles and its Analytical Application for Detection of Hydrogen Peroxide and Glucose. Analyst 2012, 137, 1706-1712.

ACS Paragon Plus Environment

33

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20. 21.

22.

23.

24.

25.

26.

27. 28.

29.

30. 31.

32.

33.

Page 34 of 37

Chen, W.; Chen, J.; Liu, A.-L.; Wang, L.-M.; Li, G.-W.; Lin, X.-H., Peroxidase-Like Activity of Cupric Oxide Nanoparticle. ChemCatChem 2011, 3, 1151-1154. Lousada, C. M.; Johansson, A. J.; Brinck, T.; Jonsson, M., Mechanism of H2O2 Decomposition on Transition Metal Oxide Surfaces. J. Phys. Chem. C. 2012, 116, 95339543. Singh, M.; Weerathunge, P.; Liyanage, P. D.; Mayes, E.; Ramanathan, R.; Bansal, V., Competitive Inhibition of the Enzyme-Mimic Activity of Gd-Based Nanorods toward Highly Specific Colorimetric Sensing of L-Cysteine. Langmuir 2017, 33, 10006-10015. Wu, C.-W.; Harroun, S. G.; Lien, C.-W.; Chang, H.-T.; Unnikrishnan, B.; Lai, I. P.-J.; Chang, J.-Y.; Huang, C.-C., Self-Templated Formation of Aptamer-Functionalized Copper Oxide Nanorods with Intrinsic Peroxidase Catalytic Activity for Protein and Tumor Cell Detection. Sens. Actuators B. 2016, 227, 100-107. Tao, Y.; Ju, E.; Ren, J.; Qu, X., Bifunctionalized Mesoporous Silica‐Supported Gold Nanoparticles: Intrinsic Oxidase and Peroxidase Catalytic Activities for Antibacterial Applications. Adv. Mater. 2015, 27, 1097-1104. Zhang, D.; Zhao, Y.-X.; Gao, Y.-J.; Gao, F.-P.; Fan, Y.-S.; Li, X.-J.; Duan, Z.-Y.; Wang, H., Anti-Bacterial and in vivo Tumor Treatment by Reactive Oxygen Species Generated by Magnetic Nanoparticles. J. Mater. Chem. B. 2013, 1, 5100-5107. Gao, L.; Giglio, K. M.; Nelson, J. L.; Sondermann, H.; Travis, A. J., Ferromagnetic Nanoparticles with Peroxidase-Like Activity Enhance the Cleavage of Biological Macromolecules for Biofilm Elimination. Nanoscale 2014, 6, 2588-2593. Sun, H.; Gao, N.; Dong, K.; Ren, J.; Qu, X., Graphene Quantum Dots-Band-Aids used for Wound Disinfection. ACS Nano 2014, 8, 6202-6210. Natalio, F.; André, R.; Hartog, A. F.; Stoll, B.; Jochum, K. P.; Wever, R.; Tremel, W., Vanadium Pentoxide Nanoparticles Mimic Vanadium Haloperoxidases and Thwart Biofilm Formation. Nat. Nanotechnol. 2012, 7, 530-535. Wang, W.; Li, G.; Xia, D.; An, T.; Zhao, H.; Wong, P. K., Photocatalytic Nanomaterials for Solar-Driven Bacterial Inactivation: Recent Progress and Challenges. Environ. Sci.: Nano 2017, 4, 782-799. Velema, W. A.; Van Der Berg, J. P.; Hansen, M. J.; Szymanski, W.; Driessen, A. J.; Feringa, B. L., Optical Control of Antibacterial Activity. Nat. Chem. 2013, 5, 924-928. Hu, Y.; Zou, W.; Julita, V.; Ramanathan, R.; Tabor, R. F.; Nixon-Luke, R.; Bryant, G.; Bansal, V.; Wilkinson, B. L., Photomodulation of Bacterial Growth and Biofilm Formation using Carbohydrate-Based Surfactants. Chem. Sci. 2016, 7, 6628-6634. Hu, Y.; Marlow, J. B.; Ramanathan, R.; Zou, W.; Tiew, H. G.; Pottage, M. J.; Bansal, V.; Tabor, R. F.; Wilkinson, B. L., Synthesis and Properties of Photoswitchable Carbohydrate Fluorosurfactants. Aust. J. Chem. 2016, 68, 1880-1884. Anderson, S. R.; Mohammadtaheri, M.; Kumar, D.; O'Mullane, A. P.; Field, M. R.; Ramanathan, R.; Bansal, V., Robust Nanostructured Silver and Copper Fabrics with Localized Surface Plasmon Resonance Property for Effective Visible Light Induced Reductive Catalysis. Adv. Mater. Interfaces 2016, 3, Article no. 1500632.

ACS Paragon Plus Environment

34

Page 35 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45. 46. 47.

Bechet, D.; Couleaud, P.; Frochot, C.; Viriot, M.-L.; Guillemin, F.; Barberi-Heyob, M., Nanoparticles as Vehicles for Delivery of Photodynamic Therapy Agents. Trends Biotechnol. 2008, 26, 612-621. La, D. D.; Ramanathan, R.; Rananaware, A.; Bansal, V.; Bhosale, S. V., Nanostructured Charge Transfer Complex of CuTCNQF4 for Efficient Photo-Removal of Hexavalent Chromium. RSC Adv. 2016, 6, 33931-33936. Li, Y.; Zhang, W.; Niu, J.; Chen, Y., Mechanism of Photogenerated Reactive Oxygen Species and Correlation with the Antibacterial Properties of Engineered Metal-Oxide Nanoparticles. ACS Nano 2012, 6, 5164-5173. Liu, C.; Kong, D.; Hsu, P.-C.; Yuan, H.; Lee, H.-W.; Liu, Y.; Wang, H.; Wang, S.; Yan, K.; Lin, D., Rapid Water Disinfection using Vertically Aligned MoS2 Nanofilms and Visible Light. Nat. Nanotechnol. 2016, 11, 1098-1104. Pearson, A.; Zheng, H.; Kalantar-zadeh, K.; Bhargava, S. K.; Bansal, V., Decoration of TiO2 Nanotubes with Metal Nanoparticles using Polyoxometalate as a UV-Switchable Reducing Agent for Enhanced Visible and Solar Light Photocatalysis. Langmuir 2012, 28, 14470-14475. Ramanathan, R.; Bansal, V., Ionic Liquid Mediated Synthesis of Nitrogen, Carbon and Fluorine-Codoped Rutile TiO2 Nanorods for Improved UV and Visible Light Photocatalysis. RSC Adv. 2015, 5, 1424-1429. Cozzoli, P. D.; Manna, L.; Curri, M. L.; Kudera, S.; Giannini, C.; Striccoli, M.; Agostiano, A., Shape and Phase Control of Colloidal ZnSe Nanocrystals. Chem. Mater. 2005, 17, 1296-1306. Panda, A. B.; Acharya, S.; Efrima, S., Ultranarrow ZnSe Nanorods and Nanowires: Structure, Spectroscopy, and One‐Dimensional Properties. Adv. Mater. 2005, 17, 24712474. Wang, W.; Zhou, Q.; Fei, X.; He, Y.; Zhang, P.; Zhang, G.; Peng, L.; Xie, W., Synthesis of CuO Nano-and Micro-Structures and their Raman Spectroscopic Studies. CrystEngComm 2010, 12, 2232-2237. Yao, W.-T.; Yu, S.-H.; Zhou, Y.; Jiang, J.; Wu, Q.-S.; Zhang, L.; Jiang, J., Formation of Uniform CuO Nanorods by Spontaneous Aggregation: Selective Synthesis of CuO, Cu2O, and Cu Nanoparticles by a Solid−Liquid Phase ARC Discharge Process. J. Phys. Chem. B. 2005, 109, 14011-14016. Khan, M. M.; Ansari, S. A.; Pradhan, D.; Ansari, M. O.; Lee, J.; Cho, M. H., Band Gap Engineered TiO2 Nanoparticles for Visible Light Induced Photoelectrochemical and Photocatalytic Studies. J. Mater. Chem. A. 2014, 2, 637-644. Asahi, R.; Morikawa, T.; Ohwaki, T.; Aoki, K.; Taga, Y., Visible-light Photocatalysis in Nitrogen-Doped Titanium Oxides. Science 2001, 293, 269-271. Hou, W.; Cronin, S. B., A Review of Surface Plasmon Resonance‐Enhanced Photocatalysis. Adv. Funct. Mater. 2013, 23, 1612-1619. El-Trass, A.; ElShamy, H.; El-Mehasseb, I.; El-Kemary, M., CuO Nanoparticles: Synthesis, Characterization, Optical Properties and Interaction with Amino Acids. Appl. Surf. Sci. 2012, 258, 2997-3001.

ACS Paragon Plus Environment

35

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

48.

49.

50.

51.

52.

53.

54.

55.

56. 57.

58. 59.

60.

Page 36 of 37

Septina, W.; Prabhakar, R. R.; Wick, R.; Moehl, T.; Tilley, S. D., Stabilized Solar Hydrogen Production with CuO/CdS Heterojunction Thin Film Photocathodes. Chem. Mater. 2017, 29, 1735-1743. Zhou, L.; Yu, L.; Ding, M.; Li, J.; Tan, H.; Wang, Z.; Fu, Q., Synthesis and Characterization of pH-Sensitive Biodegradable Polyurethane for Potential Drug Delivery Applications. Macromolecules 2011, 44, 857-864. Lin, H.-F.; Liao, S.-C.; Hung, S.-W., The DC Thermal Plasma Synthesis of ZnO Nanoparticles for Visible-Light Photocatalyst. J. Photochem. Photobiol. A. 2005, 174, 82-87. Ishibashi, K.-i.; Fujishima, A.; Watanabe, T.; Hashimoto, K., Quantum Yields of Active Oxidative Species Formed on TiO2 Photocatalyst. J. Photochem. Photobiol. A. 2000, 134, 139-142. Angelé-Martínez, C.; Nguyen, K. V. T.; Ameer, F. S.; Anker, J. N.; Brumaghim, J. L., Reactive Oxygen Species Generation by Copper(II) Oxide Nanoparticles Determined by DNA Damage Assays and EPR Spectroscopy. Nanotoxicol. 2017, 11, 278-288. Sharma, A.; Dutta, R. K., Studies on the Drastic Improvement of Photocatalytic Degradation of Acid Orange-74 Dye by TPPO Capped CuO Nanoparticles in Tandem with Suitable Electron Capturing Agents. RSC Adv. 2015, 5, 43815-43823. Applerot, G.; Lellouche, J.; Lipovsky, A.; Nitzan, Y.; Lubart, R.; Gedanken, A.; Banin, E., Understanding the Antibacterial Mechanism of CuO Nanoparticles: Revealing the Route of Induced Oxidative Stress. Small 2012, 8, 3326-3337. Malka, E.; Perelshtein, I.; Lipovsky, A.; Shalom, Y.; Naparstek, L.; Perkas, N.; Patick, T.; Lubart, R.; Nitzan, Y.; Banin, E.; Gedanken, A., Eradication of Multi-Drug Resistant Bacteria by a Novel Zn-doped CuO Nanocomposite. Small 2013, 9, 4069-4076. Ravindra Kumar, S.; Imlay, J. A., How Escherichia coli Tolerates Profuse Hydrogen Peroxide Formation by a Catabolic Pathway. Journal of Bacteriol. 2013, 195, 4569-4579. Shao, H.-B.; Chu, L.-Y.; Lu, Z.-H.; Kang, C.-M., Primary Antioxidant Free Radical Scavenging and Redox Signaling Pathways in Higher Plant Cells. Int. J. Biol. Sci. 2008, 4, 8-14. Wang, L.; Hu, C.; Shao, L., The Antimicrobial Activity of Nanoparticles: Present Situation and Prospects for the Future. Int. J. Nanomedicine 2017, 12, 1227-1249. Wang, L.-S.; Gupta, A.; Duncan, B.; Ramanathan, R.; Yazdani, M.; Rotello, V. M., Biocidal and Antifouling Chlorinated Protein Films. ACS Biomater. Sci. Eng. 2016, 2, 1862-1866. Kandjani, A. E.; Griffin, M. J.; Ramanathan, R.; Ippolito, S. J.; Bhargava, S. K.; Bansal, V., A New Paradigm for Signal Processing of Raman Spectra Using a Smoothing Free Algorithm: Coupling Continuous Wavelet Transform with Signal Removal Method. J. Raman Spectrosc. 2013, 44, 608-621.

ACS Paragon Plus Environment

36

Page 37 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

Table of Contents Graphic

ACS Paragon Plus Environment

37